The present invention relates to in situ immunodetection of antigens.
Numerous devices and methods are known for detecting antigens ex situ, i.e., on or within tissue, fluid or other samples that have been removed from the body. In a typical protocol antibodies may be raised against one or more epitopes on a target protein by injecting the protein into a rabbit, human or other host, allowing the host to produce antibodies against the protein, and then removing and purifying sera from the host. The antibody is then brought into contact with the sample under circumstances which are favorable for the formation of an antibody/antigen complex. In cases where the signal is low, such as where the amount of antigen is low or the antigen/antibody binding is poor, one or more amplification steps may be used. Typically, a visible chromophore, radioactive, fluorescent, oligonucleotide or other marker is attached to the antibody or to one of the amplification reagents to facilitate detection of the antigen complex. The terms "label" and "marker" are used herein interchangeably.
Antibodies used in such methods may be more or less specifically selective for the target antigen, and may bind with a greater or lesser efficiency to the target antigen. Typically, useful antibodies will have a good specificity for the target antigen and an association constant (ka) of approximately 10.sup.3 -10.sup.9 with the target antigen. Antibodies may also be polyclonal or monoclonal, or some combination of the two.
A known method of immunodetection designed by Charles Cantor uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.
Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology (2). The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
Still other methods may utilize synthetic antibodies such as those available through molecular imprinting technology (see, e.g., U.S. Pat. No. 5,110,833 to Mosbach). As used herein, the terms "antibody" and "antibodies" include synthetic antibodies and other antibody analogs.
While these and other known methods of immunodetection have been applied to nonliving materials, and to living cells ex situ (e.g., tumor cells (3), they have never to our knowledge been applied in situ. As used herein in situ refers to tissues, cells, fluids and other body components that are still coupled to the body during the antibody binding step, as opposed to having been biopsied or otherwise removed from the body for the antigen binding step.
Application of antigen detecting methods to in situ body components is highly desirable. For example, such methods can be performed outside the confines of a laboratory or clinical setting, without the taking of tissue samples. Where the results are visualized macroscopically, the methods can also be performed without the need for specialized equipment such as microscopes or plate readers (4). In situ detection of antigens on a body surface such as the skin of a patient, for example, can facilitate field screening of numerous pathogenic agents such as virus, bacteria, fungi and mycoplasma appearing on the skin surface, and assist in the diagnosis of skin conditions such as psoriasis. In situ detection of antigens may also be used to detect antigens which are present below the surface of the skin, such as where the detection takes place in an open wound or an incision, or in a body cavity. In these and other instances, antigens are said to be proximally associated with a body component if they can be detected with application of one or more of the devices and methods disclosed herein to the body component.
Due to the presence of Langerhans and other specialized cells in the skin, in situ immunodetection of antigens may also be useful in the diagnosis and prognostication of systemic diseases including AIDS and some cancers. Langerhans are professional antigen presenting cells (APCs) which comprise approximately 2% of the human skin cell population. They function both as the skin's antigen presenting cells and as depositories for foreign antigenic fragments, accumulating and presenting at the skin surface circulating antigens characteristic of a broad range of localized and systemic conditions. In the case of AIDS, for example, it is known that human immuno-deficiency virus can infect Langerhans cells, (5), and that resident Langerhans cells can contain the HIV-1 gag protein and have viral particles budding from their membranes(6,7). Proviral DNA(5) and tat, rev, nef and env mRNA(8) have been demonstrated in Langerhans cells, and HIV-specific nucleic acids have been detected on the surface of biopsied skin specimens using PCR (8).
Of course, in situ immunodetection of disease conditions need not necessarily rely on Langerhans cells. Keratinocytes, for example, are epidermal cells which are primary targets of Human Papilloma Virus (HPV), and a topical test for HPV antigens could be developed in accordance with the devices and methods disclosed herein.
Thus, there is a need to provide devices and methods for in situ immunodetection of antigens.